Compositions and methods for inhibiting inflammation and diseases using an alginic acid-based antimicrobial compound

ABSTRACT

The present disclosure relates to compositions and methods for inhibiting inflammation and reducing the risk of spreading sexually transmitted diseases using an alginic acid-based antimicrobial compound. Such compositions provide dual protection by (1) attacking and inactivating viruses and other microbes and (2) blocking the host response that viruses trigger to invade host cells. Such compositions can also be part of an acid buffering contraceptive.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/106,755, filed on Jun. 20, 2016, which is the national phase entry ofInternational application number PCT/US14/71664, filed Dec. 19, 2014,which claims benefit to U.S. provisional application No. 61/918,445,filed on Dec. 19, 2013, the contents of which are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present disclosure relates to compositions and methods forinhibiting inflammation and diseases, such as sexually transmitteddiseases, using an alginic acid-based antimicrobial compound. Suchcompositions provide dual protection by (1) attacking and inactivatingviruses and other microbes and (2) blocking the host response thatviruses trigger to invade host cells. Such compositions can also be partof an acid buffering contraceptive.

BACKGROUND OF INVENTION

Sexually transmitted diseases (STDs) are illnesses that are transmittedbetween humans through sexual behavior. STDs can be caused by bacteria,viruses, protozoa, and parasites. While many STDs, especially thosecaused by bacteria, protozoa, and parasites can be cured withantibiotics or other medication, most STDs caused by viruses cannot becured. Rather, drugs are used to keep the viral levels below levelswhich cause symptoms. Of the known viral STDs, the three mostproblematic are human immunodeficiency virus, hepatitis virus, andherpes simplex virus.

Human immunodeficiency virus (HIV), the etiologic agent of acquiredimmunodeficiency syndrome (AIDS) is the fastest growing cause of deathin women of reproductive age. Worldwide, the heterosexual transmissionof AIDS is the prevalent mode of transmission of AIDS, accounting forabout 90% of all HIV infections in women. Therefore, significantattention has been directed to investigating measures that block sexualspreading of HIV infection. As there is no effective treatment orvaccine against AIDS, preventive measures are the primary tools that canpresently reduce transmission of HIV. For example, the consistent andcorrect use of condoms represents an effective barrier to prevent HIVtransmission. However, the risk of acquiring infection can only besignificantly reduced if condoms are used for almost all sexualintercourse in HIV prevalent communities; a result that can not beachieved despite intensive prevention programs to increase condom use.

Hepatitis viruses cause inflammation of the liver. The most commonhepatitis viruses are the five unrelated hepatotropic viruses, HepatitisA (HAV), Hepatitis B (HBV), Hepatitis C (HCV), Hepatitis D (HDV), andHepatitis E (HEV). The five hepatitis viruses are transmitted via eithera fecal-oral route (hepatitis A and E) or via blood contact (hepatitisB, C, and D). HAV, HBV, and HEV can generally be cleared by the body'simmune system. However, HCV and HBV can cause chronic hepatitis. HDV isunique in that it can only propagate in the presence of HBV. However,the presence of HDV exacerbates the symptoms of HBV. Proper condom usecan help to prevent the transmission of HBV, HCV, and HDV.

Herpes simplex virus (HSV) belongs to a family of viruses, nine of whichare known to cause diseases in humans. The nine human herpes virusescause a variety of diseases including chicken pox, shingles,mononucleosis, sixth disease, and Kaposi's sarcoma. The HSV familyincludes two viruses, referred to as HSV-1 and HSV-2, that causeblisters in the skin or mucous membranes of the mouth, lips and/orgenitals. While both viruses can infect either the mouth or genitals,HSV-1 predominantly infects orofacial tissue, whereas HSV-2predominantly infects genital tissue. Both HSV-1 and HSV-2 aretransmitted through close physical contact. The Center for DiseaseControl (CDC) estimates that one in six people aged 14-49 are infectedwith HSV-2. While condom use can reduce the risk of spreading HSV,however, HSV can still be transmitted via contact with genital skin thatis not covered by the condom.

Significant emphasis has been placed on the development of intravaginalmicrobicidal agents capable of preventing and/or reducing the spread ofa variety of STDs. The development of microbicides for topical userepresents an important alternative to condom usage. A microbicide isany agent that kills or deactivates disease-causing microbes, includingviruses. According to the International Association of Physicians inAIDS CARE (IAPAC), the definition of microbicides also includesinterventions that can block or prevent infection, as well asamplification of the body's natural defenses to prevent infectionthrough sexual acts.

Ideally, microbicides should have little or no side effects at aneffective microbicidal concentration. Accordingly, the drug used as amicrobicide should have little or no immunosuppressive activity at aneffective microbicidal concentration. In addition, the ideal microbicideshould sufficiently withstand varying temperatures and acceptablyfunction within varied pH ranges (ranges of alkaline and acidic levelsin the vagina). Further, it should not eliminate the natural beneficiallactobacilli that reside in the vagina and contribute to vaginal health.

Topical microbicides would be even more beneficial if they also hadcontraceptive capabilities. Contraception is also important for womenwith STDs to prevent transmitting diseases to future generation,especially since many women with STDs are of childbearing age. Atpresent, a majority of commercially available dual-purpose spermicidalmicrobicides have detergent ingredients that disrupt cell membranes. Themost widely used vaginal spermicide, nonoxynol-9 (N-9), because of itsmembrane disruptive properties, has been shown to damage thecervicovaginal epithelium, cause an acute inflammatory tissue response,alter vaginal microflora, and enhance the risk of promotingopportunistic infections in the genitourinary tract. N-9 is also toxicto vaginal and cervical cells which increases the permeability ofvaginal tissue. It can also kill the Lactobacillus sp. that populate thevaginal tract and are generally regarded as beneficial. Lactobacillusproduce lactic acid and hydrogen peroxide, which helps maintain theacidic pH of the vagina (˜pH 3.5 to 5.0) and a healthy vaginal flora. Atthis pH, a number of STD-causing organisms like HIV are inactivated.

Other microbicides in the form of vaginal creams and ointments arecurrently available over the counter or by prescription. Still othersare in various stages of development. Examples include octoxynol-9 andbenzalkonium chloride. Gels designed to control vaginal pH are alsoavailable, such as AciJel™ (Ortho-McNeil Pharmaceutical Corp., Raritan,N.J.) which is a water dispersible buffered gel having a pH of 3.9 to4.1. It is used to restore and maintain normal vaginal acidity. Suchgels are designed to control vaginal pH and are not specificallydesigned to prevent STDs and/or contraception, and thus do not alwayspossess effective microbicidal activity.

As discussed, presently marketed vaginal contraceptive compositions,often containing N-9 as an active ingredient, are generally known in theart. While presently marketed vaginal contraceptive formulations aid inpreventing pregnancy, their ability to effectively prevent STDs,particularly HIV/AIDS, is very limited. Moreover, recent analyses showthat N-9, when used frequently by women at high risk may actuallyincrease the risk of HIV infection (WHO 2002, WHO/CONRAD technicalconsultation on nonoxynol-9, Geneva).

Additionally, several microbicides under development containanti-retroviral agents that had originally been developed for thetreatment of patients with HIV infection. However, only temporary andlimited benefits are observed in HIV-infected patients treated with anyof the actual anti-retrovirals or combinations thereof. The limitedability of these agents to decrease viral burden, the rapid developmentof resistance and the toxic side-effects of most drugs has limited theirlong-term efficacy. One major problem associated with the administrationof antiviral agents to patients is their poor ability to penetrate andtarget infected cells. Rapid drug clearance and the toxicity of parentcompounds or metabolites also constitute some of the major drawbacksthat may slow down the development and use of many antiviral agents.Given the severe toxicity of antiviral agents actually available totreat AIDS and other viral diseases and their limited ability to targetinfected cells, strategies aimed at reaching therapeutic levels of drugsinto infected cells and reducing toxicity is needed.

Recent studies have shown that a significant contribution to theantimicrobial properties naturally present in the vagina is primarilydue to the microbicidal activity of the lactic acid molecule, and is notnecessarily due to low pH alone or to the presence of hydrogen peroxide.(O'Hanlon et al., BMC Infect Dis., 11:200, 2011). In particular, it hasbeen shown that in vaginal fluid, bacteria associated with bacterialvaginosis can be suppressed with lactic acid, but to a much lesserextent with other acids at the same pH.

Accordingly, there is a need for an alternative easy to use STDinhibitor that effectively reduces the risk of transmission ofviral-based STDs. Such compositions should be useful for vaginaladministration in effective doses that do not inactivate Lactobacillussp. or cause overt vaginal irritation or other toxicity. Thecompositions would be even more beneficial if they also hadcontraceptive capabilities.

SUMMARY OF INVENTION

The embodiments disclosed below satisfy this need. The followingsimplified summary is provided in order to establish a basicunderstanding of some aspects of the claimed subject matter. Thissummary is not an extensive overview, and is not intended to identifykey/critical elements or to delineate the scope of the claimed subjectmatter.

One embodiment is a composition for inhibiting the transmission ofdiseases. The composition includes1-(6-aminopurin-9-yl)propan-2-yloxymethylphosphonic acid (tenofovir) ora physiologically functional derivative thereof, alginic acid polymer,and an aqueous based carrier. An exemplary alginic acid polymer of thepresent disclosure have an average molecular weight between about 20,000to about 400,000 and a ratio of mannuronate:guluronate residues in thepolymer between about 0.25 to about 2.0. In another embodiment the ratioof mannuronate:guluronate residues in the alginic acid polymer isbetween 0.3 to 1.5. In another embodiment the ratio ofmannuronate:guluronate residues in the alginic acid polymer is between0.3 to 1. In another embodiment the ratio of mannuronate:guluronateresidues in the alginic acid polymer is between 0.3 to 0.8. In anotherembodiment the ratio of mannuronate:guluronate residues in the alginicacid polymer is between 0.3 to 0.6. In another embodiment, the ratio ofmannuronate:guluronate residues in the alginic acid polymer is about0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8,0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45,1.5, 1.55, 1.6, 1.65 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2.0. In anotherembodiment, the average molecular weight of the alginic acid is between75,000 to 375,000. In another embodiment, the average molecular weightof the alginic acid is between 100,000 to 300,000. In another embodimentthe average molecular weight of the alginic acid is between 100,000 to200,000. In another embodiment, the average molecular weight of thealginic acid is between 125,000 to 175,000. In another embodiment, theaverage molecular weight of the alginic acid is at least 20,000. Inanother embodiment, the average molecular weight of the alginic acid isat least 50,000. In another embodiment, the average molecular weight ofthe alginic acid is at least 75,000. In another embodiment, the averagemolecular weight of the alginic acid is at least 100,000. In anotherembodiment, the average molecular weight of the alginic acid is at least125,000. In another embodiment, the average molecular weight of thealginic acid is at least 150,000.

In another embodiment, the composition also includes lactic acid. Inanother embodiment, the lactic acid is the “L” form of lactic acid. Inanother embodiment, the composition also includes pamoic acid or a saltor ester thereof. In yet another embodiment, the composition alsoincludes carrageenan. In yet another embodiment, the carrageenan is iotacarrageenan. In yet another embodiment, the composition may also includea buffering agent (such as citric acid, potassium acid tartrate, sorbicacid, fumaric acid, ascorbic acid, stearic acid, oleic acid, tartaricacid, potassium bitartrate, edetic acid ethylenediaminetetracetic acid,acetic acid, malic acid, and the like), thickener (such as xanthan gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodiumcarboxymethyl cellulose, chitosan, polycarbophil, carbopol, gellan gum,poloxamer, carrageenan, iota carrageenan, and the like), humectant (suchas glycerol, polyethylene glycols, propylene glycols, sorbitol,triacetin, and the like), or preservative (such as benzoic acid, sodiumbenzoate, methylparaben, ethylparaben, butylparaben, propylparaben,benzylalkonium chloride, phenylmercuric nitrate, chlorhexidine, and thelike). In yet another embodiment, the carrier of the composition iswater.

Another embodiment is an acid buffering contraceptive. The acidbuffering contraceptive includes alginic acid, a bioadhesive compound,and lactic acid in a pharmaceutically acceptable carrier. The alginicacid has an average molecular weight between 20,000 to 400,000 and aratio of mannuronate:guluronate residues between 0.25 to 2.0. In anotherembodiment the mannuronate:guluronate residues is between 0.3 to 1.5. Inanother embodiment the mannuronate:guluronate residues is between 0.3to 1. In another embodiment, the mannuronate:guluronate residues isbetween 0.3 to 0.8. In another embodiment, the mannuronate:guluronateresidues is between 0.3 to 0.6. In another embodiment, the ratio ofmannuronate:guluronate residues in the alginic acid polymer is about0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8,0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45,1.5, 1.55, 1.6, 1.65 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2.0. In anotherembodiment, the average molecular weight of the alginic acid is between75,000 to 375,000. In another embodiment, the average molecular weightof the alginic acid is between 100,000 to 300,000. In another embodimentthe average molecular weight of the alginic acid is between 100,000 to200,000. In another embodiment, the average molecular weight of thealginic acid is at least 50,000. In another embodiment, the averagemolecular weight of the alginic acid is at least 75,000. In anotherembodiment, the average molecular weight of the alginic acid is at least100,000. In another embodiment, the average molecular weight of thealginic acid is at least 125,000. In another embodiment, the averagemolecular weight of the alginic acid is at least 150,000. Using theabove described alginic acids may further enhance the ability of theacid buffering contraceptive to inhibit the transmission of diseases.

In one embodiment, the bioadhesive compound of the acid bufferingcontraceptive may be xanthan gum, carrageenan, iota carrageenan,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodiumcarboxymethyl cellulose, chitosan, polycarbophil, carbopol, and thelike. In another embodiment, the acid buffering compound may alsoinclude buffering agents (such as citric acid, potassium acid tartrate,sorbic acid, fumaric acid, ascorbic acid, stearic acid, oleic acid,tartaric acid, potassium bitartrate, edetic acidethylenediaminetetracetic acid, acetic acid, malic acid, and the like),thickeners (such as xanthan gum, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, chitosan,polycarbophil, carbopol, gellan gum, poloxamer, carrageenan, iotacarrageenan, and the like), humectants (such as glycerol, polyethyleneglycols, propylene glycols, sorbitol, triacetin, and the like),preservatives (such as benzoic acid, sodium benzoate, methylparaben,ethylparaben, butylparaben, propylparaben, benzylalkonium chloride,phenylmercuric nitrate, chlorhexidine, and the like), or agents thatenhance drug solubility, permeability and absorption (such as pamoicacid and salts and esters thereof). In another embodiment, the lacticacid is L-lactic acid.

In one embodiment, the acid buffering contraceptive includes alginicacid, xanthan gum, and lactic acid. In another embodiment, the acidbuffering contraceptive includes alginic acid, carrageenan, and lacticacid. In another embodiment, the acid buffering contraceptive includesalgininc acid, iota carrageenan, and lactic acid. In yet anotherembodiment, the acid buffering contraceptive includes alginic acid,xanthan gum, pamoic acid, and lactic acid. In another embodiment, theacid buffering contraceptive includes alginic acid, carrageenan, pamoicacid, and lactic acid. In another embodiment, the acid bufferingcontraceptive includes algininc acid, iota carrageenan, pamoic acid, andlactic acid.

In another embodiment, the acid buffering contraceptive may also includea buffering agent (such as citric acid, potassium acid tartrate, benzoicacid, sorbic acid, fumaric acid, ascorbic acid, stearic acid, oleicacid, tartaric acid, potassium bitartrate, edetic acidethylenediaminetetracetic acid, acetic acid, malic acid, and the like),thickener (such as xanthan gum, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, chitosan,polycarbophil, carbopol, gellan gum, poloxamer, carrageenan, iotacarrageenan, and the like), humectant (such as glycerol, polyethyleneglycols, propylene glycols, sorbitol, triacetin, and the like), orpreservative (such as benzoic acid, sodium benzoate, methylparaben,ethylparaben, butylparaben, propylparaben, benzylalkonium chloride,phenylmercuric nitrate, chlorhexidine, and the like). In yet anotherembodiment, the carrier of the composition is water.

Another embodiment is an acid buffering contraceptive. The acidbuffering contraceptive includes1-(6-aminopurin-9-yl)propan-2-yloxymethylphosphonic acid (tenofovir) ora physiologically functional derivative thereof, alginic acid, abioadhesive compound, and lactic acid in a pharmaceutically acceptablecarrier. The alginic acid has an average molecular weight between 20,000to 400,000 and a ratio of mannuronate:guluronate residues between 0.25to 2.0. In another embodiment the mannuronate:guluronate residues isbetween 0.3 to 1.5. In another embodiment the mannuronate:guluronateresidues is between 0.3 to 1. In another embodiment, themannuronate:guluronate residues is between 0.3 to 0.8. In anotherembodiment, the mannuronate:guluronate residues is between 0.3 to 0.6.In another embodiment, the ratio of mannuronate:guluronate residues inthe alginic acid polymer is about 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15,1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65 1.7, 1.75, 1.8,1.85, 1.9, 1.95, or 2.0. In another embodiment, the average molecularweight of the alginic acid is between 75,000 to 375,000. In anotherembodiment, the average molecular weight of the alginic acid is between100,000 to 300,000. In another embodiment the average molecular weightof the alginic acid is between 100,000 to 200,000. In anotherembodiment, the average molecular weight of the alginic acid is at least50,000. In another embodiment, the average molecular weight of thealginic acid is at least 75,000. In another embodiment, the averagemolecular weight of the alginic acid is at least 100,000. In anotherembodiment, the average molecular weight of the alginic acid is at least125,000. In another embodiment, the average molecular weight of thealginic acid is at least 150,000. Using the above described alginicacids may further enhance the ability of the acid bufferingcontraceptive to inhibit the transmission of diseases.

In one embodiment, the bioadhesive compound of the acid bufferingcontraceptive with tenofovir may be xanthan gum, carrageenan, iotacarrageenan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,sodium carboxymethyl cellulose, chitosan, polycarbophil, carbopol, andthe like. In another embodiment, the acid buffering compound may alsoinclude buffering agents (such as citric acid, potassium acid tartrate,sorbic acid, fumaric acid, ascorbic acid, stearic acid, oleic acid,tartaric acid, potassium bitartrate, edetic acidethylenediaminetetracetic acid, acetic acid, malic acid, and the like),thickeners (such as xanthan gum, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, chitosan,polycarbophil, carbopol, gellan gum, poloxamer, carrageenan, iotacarrageenan, and the like), humectants (such as glycerol, polyethyleneglycols, propylene glycols, sorbitol, triacetin, and the like),preservatives (such as benzoic acid, sodium benzoate, methylparaben,ethylparaben, butylparaben, propylparaben, benzylalkonium chloride,phenylmercuric nitrate, chlorhexidine, and the like), or agents thatenhance drug solubility, permeability and absorption (such as pamoicacid and salts and esters thereof). In another embodiment, the lacticacid is L-lactic acid.

In one embodiment, the acid buffering contraceptive with tenofovirincludes alginic acid, xanthan gum, and lactic acid. In anotherembodiment, the acid buffering contraceptive includes alginic acid,carrageenan, and lactic acid. In another embodiment, the acid bufferingcontraceptive includes algininc acid, iota carrageenan, and lactic acid.In yet another embodiment, the acid buffering contraceptive includesalginic acid, xanthan gum, pamoic acid, and lactic acid. In anotherembodiment, the acid buffering contraceptive includes alginic acid,carrageenan, pamoic acid, and lactic acid. In another embodiment, theacid buffering contraceptive includes algininc acid, iota carrageenan,pamoic acid, and lactic acid.

In another embodiment, the acid buffering contraceptive with tenofovirmay also include a buffering agent (such as citric acid, potassium acidtartrate, benzoic acid, sorbic acid, fumaric acid, ascorbic acid,stearic acid, oleic acid, tartaric acid, potassium bitartrate, edeticacid ethylenediaminetetracetic acid, acetic acid, malic acid, and thelike), thickener (such as xanthan gum, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose,chitosan, polycarbophil, carbopol, gellan gum, poloxamer, carrageenan,iota carrageenan, and the like), humectant (such as glycerol,polyethylene glycols, propylene glycols, sorbitol, triacetin, and thelike), or preservative (such as benzoic acid, sodium benzoate,methylparaben, ethylparaben, butylparaben, propylparaben, benzylalkoniumchloride, phenylmercuric nitrate, chlorhexidine, and the like). In yetanother embodiment, the carrier of the composition is water.

Another embodiment is a method for reducing the risk of spreading asexually transmitted disease by topically administering an effectiveamount of any of the compositions described above and herein. In anotherembodiment, the compositions may be topically applied to the vagina,cervix, mouth, anus, and/or rectum. The compositions may be appliedbefore sexual activity. In one embodiment, the compositions may beapplied at least 15 minutes or at least 30 minutes or at least 1 hour orat least 1.5 hours or at least 2 hours or at least 2.5 hours or at least3 hours or at least 3.5 hours or at least 4 hours or at least 4.5 hoursor at least 5 hours or at least 6 hour or at least 7 hours or at least 8hours or at least 9 hours or at least 10 hours or at least or at least12 hours prior to sexual activity. The compositions may also be appliedafter sexual activity. In one embodiment, the compositions may beapplied immediately after or within 5 minutes after or within 10 minutesafter or within 15 minutes after or within 20 minutes after or within 30minutes after or within 45 minutes after or within 1 hour after, orwithin 2 hours after or within 3 hours after or within 4 hours after orwithin 5 hours after or within 6 hours after or within 7 hours after orwithin 8 hours after or within 9 hours after or within 10 hours aftersexual activity. In another embodiment, the compositions may be appliedto the surface of a physical barrier device such as a condom, sponge, ordiaphragm before using the barrier device.

In another embodiment are methods for manufacturing the compositionsdescribed herein. In one embodiment, the1-(6-aminopurin-9-yl)propan-2-yloxymethylphosphonic acid (tenofovir) ora physiologically functional derivative thereof is dissolved in a basicsolution. Once the tenofovir is dissolved, the solution is brought tobelow neutral and alginic acid is added. In one embodiment, the pH ofthe solution is reduced with the addition of lactic acid. In anotherembodiment, additional additives are added based on the pH of theadditive. For example, buffering agents may be used to reduce the pH ofthe solution, i.e. make it more acidic. In another embodiment,thickeners may be added. In some methods, thickeners may be added lastsince the addition of thickeners may inhibit the incorporation of someingredients.

In another embodiment are kits containing the above describedcompositions in an easy to dispense container. Non-limiting examples ofdispensing containers include a bottle, tube, a syringe, a suppository,or a pump. The kit may also contain a device for applying thecompositions to the desired surface and/or instructions for use orapplication. Non-limiting applicator devices include a syringe, asponge, a brush, a swab, or a spatula. In one embodiment, thecompositions may be portioned out in single use doses. Non-limitingexamples include pre-filled syringes, pre-filled squeeze tubes, orsuppositories. In another embodiment, the composition may be coated onbarrier devices. Non-limiting examples of barrier devices includesponges, condoms or diaphragms.

Other aspects of the disclosure are found throughout the specification.

DETAILED DESCRIPTION OF INVENTION

Disclosed herein are compositions and methods for inhibitinginflammation and reducing the risk of spreading sexually transmitteddiseases (STDs) using an alginic acid-based antimicrobial compounds.Such compositions provide a dual protection by (1) attacking andinactivating viruses and other microbes and (2) blocking the hostresponse that viruses activate to facilitate host cell invasion. Morespecifically, the compositions and methods disclosed herein relate tosynergistic compositions comprising a combination of alginic acid and anantiviral agent in a carrier that enhances the efficacy of theindividual components. The alginic acid-based antimicrobial compoundscan also be part of an acid buffering contraceptive.

To facilitate understanding of the disclosure that follows, a number ofterms are defined below.

When the terms “one,” “a,” or “an” are used in this disclosure, theymean “at least one” or “one or more,” unless otherwise indicated.

As used herein, the terms “antimicrobial,” “microbicide,” and“microbicidal” refer to a compound capable of preventing or inhibitingthe growth and/or preventing or reducing the infectivity of microbes,including viruses, bacteria, fungi, protozoa, parasites, and algae.

As used herein, the term “sexually transmitted disease” is usedinterchangeably with “STD,” “sexually transmitted infection,” “STI”and/or the plural thereof. An STD is an illness or pathophysiologicalcondition that has a significant probability of transmission betweenhumans by means of any form of sexual contact, including kissing. Theterm STD may also encompass a person who is infected, and maypotentially infect others, without showing signs of disease orinfection.

The terms “synergy” and “synergistic” mean that the effect achieved withthe compounds used together is greater than the sum of the effects thatresults from using the compounds separately, i.e. greater than whatwould be predicted based on the two active ingredients administeredseparately. A synergistic effect may be attained when the compounds are:(1) co-formulated and administered or delivered simultaneously in acombined formulation; (2) delivered by alternation or in parallel asseparate formulations; or (3) by some other regimen. A synergisticantiviral effect denotes an antiviral effect which is greater than thepredicted purely additive effects of the individual compounds of thecombination.

As used herein, the term “physiologically functional derivative” refersto a pharmaceutically active compound with equivalent or near equivalentphysiological functionality to tenofovir when administered incombination with another pharmaceutically active compound in acombination of the disclosure. As used herein, the term “physiologicallyfunctional derivative” includes any: physiologically acceptable salt,ether, ester, prodrug, solvate, stereoisomer including enantiomer,diastereomer or stereoisomerically enriched or racemic mixture, and anyother compound which upon administration to the recipient, is capable ofproviding (directly or indirectly) such a compound or anantiviral-active metabolite or residue thereof.

As used herein, the term “contacting” refers to any suitable method ofbringing one or more of the alginic acid-based compounds describedherein into contact with a sexually-transmitted or sexually-acquiredmicrobe or microbial cell, as described herein. In vitro or ex vivo,this is achieved by exposing the microbe or microbial cell to themicrobicide in a suitable medium. For exemplary in vivo applications,topical methods of administration are suitable as described herein.

As used herein, the term “matrix” is meant to refer to a plurality ofdifferent molecules that form a three-dimensional structure via ionicinteractions there between.

The term “buffering capabilities” means the ability to maintain adesired pH when contacted with a compound having a different pH. Inparticular, buffering capabilities means the ability to maintain ahealthy vaginal pH.

The term “contacted with ejaculate” means the presence of semen in thevolume normally occurring during ejaculation, e.g., between 0.1 to 11milliliters (Rehan, et al., Fertil Steril. 1975, 26:492-502).

The term “basic solution” means a solution that has a pH above 7, suchas 8, 9, 10, 11, 12, or 13. Exemplary bases for making a “basicsolution” include, but are not limited to sodium hydroxide, potassiumhydroxide, and lithium hydroxide.

The term “inhibiting disease” as used herein generally refers to theinhibition of sexually transmitted diseases, which includes inhibitingthe spread thereof.

The term “alginic acid” or “alginic acid polymer” or “alginate” are usedinterchangeably and refer to a polysaccharide polymer ofbeta-D-mannuronate and alpha-L-glucuronate.

The alginic acid-based microbicide compositions and methods disclosedherein prevent or reduce the risk of the transmission of STDs and/orcommon vaginal infections. STDs include, but are not limited to,HIV/AIDS, herpes (caused by herpes simplex virus type 1 (HSV-1) orherpes simplex virus type 2 (HSV-2), hepatitis, gonorrhea, chlamydia,syphilis, and trichomoniasis. Non-limiting examples of common vaginalinfections include bacterial vaginosis (BV) and vaginal candidiasis.Similar compositions and methods of application of such compositions, asdescribed herein, can be used for preventing or treating STDs and/orcommon vaginal infections. Additional chemicals may be added to thealginic acid-based microbicide compositions to form an acid bufferingcontraceptive.

The compositions of the present disclosure comprise a combination of analginic acid polymer and a particular antiviral agent, tenofovir.Alginic acid is an acidic polymer that (1) has mucosal adhesive andbioadhesive properties that provide a physical barrier, (2) hasbuffering capabilities to help maintain an acidic environment in thevagina, and (3) has anti-inflammatory properties that reduce viralinfection in host cells. Tenofovir is an antiretroviral drug designed toinhibit reverse transcriptase. The prodrug form of tenofovir, tenofovirdisproxyl fumarate, has been approved by the U.S. Food and DrugAdministration for treating HIV and chronic hepatitis B and may beeffective against other viruses such as herpes. (Andrei, et al., CellHost Microbe., 10:379-89, 2011). In exemplary embodiments, a synergisticeffect is achieved between the alginic acid polymer and antiviral agent.More particularly, the negatively charged monophosphate moiety oftenofovir forms ionic interactions with the alginic acid polymer,facilitating prolonged release of the tenofovir, thus enhancingefficacy. In other embodiments, the alginic acid polymer and tenofovircan be combined with lactic acid, and a bioadhesive such as xanthan gumor carrageenan to form an acid buffering contraceptive. The acidbuffering contraceptive forms a matrix upon contact with ejaculate thattraps sperm and other microbes present in ejaculate.

Though not wishing to be bound by any particular theory of operation, itis further believed that the compositions of the present disclosureexhibit improved efficacy because the tenofovir concentration ismaintained between effective and toxic levels, by way of alginic acid'smucoadhesive and bioadhesive properties that inhibit the dilution of theantiretroviral drug away from the delivery point, thereby improvingtargeting and localization of the drug. In this context, mucoadhesionand bioadhesion increases the intimacy and duration of contact betweenthe tenofovir and the mucosal surface. The combined effects of thisenhanced, direct drug absorption, and the decrease in excretion ratethat results from reduced diffusion and improved localizationsignificantly enhances bioavailability of the drug and allows for asmaller dosage and less frequent administration.

Microbial Infection and Inflammation

Toll-like receptors (TLRs) are the immune systems' early warning signagainst exposure to potentially harmful substances, especially chemicalsderived from microbes. Specifically, TLRs bind to molecular structurescalled pathogen-associated molecular patterns (PAMPs) that are presentin bacteria, protozoa, fungi, and viruses. Upon activation, TLRs triggermultiple biochemical cascades that activate inflammation and cellularimmune defenses, including nuclear factor kappa-light-chain-enhancer ofactivated B cells (NFkB). NFkB is the master gene regulator ofinflammation, activation of which results in the increased synthesis ofmany inflammatory mediatory and pro-inflammatory cytokines, includingTNF-alpha, IL-1, and other interleukins. For many viruses, activation ofthe inflammatory pathways that typically aid in immune defense againstmicrobial infection, instead, enhance infectivity. It is now known thatcertain viruses, including HIV, hepatitis, and HSV, require theactivation of NFkB to infect the host cell. For example, uponactivation, NFkB binds to a site on the HIV long terminal repeat tostart transcription of integrated HIV genome. (See Pande and Ramos,Curr. Med. Chem., vol. 10, no. 16, pgs. 1603-15 (2003).) In effect, thebody's own natural response is contributing to the infectivity of thevirus.

Physical Trauma and Inflammation

Many sexual activities result in physical trauma to the tissues on amicroscopic level. Upon disruption of an anatomical barrier, e.g. mucousmembranes, neutrophils are attracted by a variety of cytokines releasedby inflammatory cells such as mast cells. Neutrophils engulf damagedcells by phagocytosis and in the process, generate and release highquantities of reactive oxygen species (ROS) that are toxic to healthysurrounding cells. ROS react with cellular anti-oxidants, in particular,glutathione. Upon exposure to ROS, reduced glutathione (GSH) shifts toits oxidized state (GSSG). Increased cellular levels of oxidizedglutathione triggers activation of NFkB. As mentioned above, activationof NFkB facilitates infection of viral STDs.

Alginic Acid

Alginates are naturally occurring polymers that can be extracted frombrown algae. The polymer is made up of two sugars, 1,4-linkedβ-D-mannuronate (M) and α-L-glucuronate (G). The polymers can be made upof blocks of consecutive M residues, blocks of consecutive D residues,or alternative D and M residues. The ratio of M:G varies depending onthe source. Most commercially available alginates have a G contentbetween 14-31%, however, alginates from Laminaria hyperborean have a Gcontent of 60%. Alginates with a more defined chemical structure andphysical properties can be obtained using bacterial biosynthesis.However, bacterial alginates have an O-acetyl group at C2 and/or C3.Acetyl groups are exclusively associated with the mannuronic acidresidues with degrees of acetylation varying from 4-57%. (See Donati andPaoletti, “Material Properties of Alginates,” in Alginates: Biology andApplications: Biology and Applications, Rehm, ed., Springer DordrechtHeidelburg, London, U K, 2009, page 10 and “The History of AglinateChemistry—Bacterial,” Cyber Colloids, LTD,www.cybercolloids.net/information/technical-articles/history-alginate-chemistry-bacterial.)Acetyl groups decrease the capacity and selectivity of cation binding,increase solution viscosity, enhance water holding capacity, and protectdegradation by alginate lyases. (See Flemming and Wingender, “TheCrucial Role of Extracellular Polymeric Substances in Biofilms,” inBiofilms in Wastewater Treatment: An Interdisciplinary Approach, Wuertz,Bishop, Wilderer, eds., IWA Publishing, London, U K 2003, page 184.)Alginates are typically extracted from brown algae using alkalisolutions and salt to form alginate salts. Alginate salts can further betreated with acid to form alginic acid.

Alginates, like polysaccharides in general, are polydisperse withrespect to molecular weight. Because of this polydispersity, the“molecular weight” of an alginate is an average over the wholedistribution of molecular weights. (See Draget, et al., “Alginates FromAlgae,” Biopolymers Online, DOI: 10.1002/3527600035.bpol6008 (2005).)The molecular-weight distribution can have implications for the uses ofalginates, as low-molecular-weight fragments containing only shortG-blocks may not take part in gel-network formation and consequently donot contribute to the gel strength. The molecular weight of mostalginate salts is in the range of about 10,000 to 600,000 grams per mole(g/mol).

Alginate polymers can be cross-linked to form a hydrogel usingmonovalent and divalent cations such as calcium, sodium, and potassium.However, only the G residues are believed to participate when usingdivalent cations. Thus, the M:G ratio of the alginate affects thephysical properties of alginate hydrogels. Furthermore, gelling time andtemperature also affect gel uniformity and strength when using cations.Slow gelling rates (e.g. greater than 15 minutes) at lower temperatures(e.g. below body temperature) result in gels with more uniformstructures and greater mechanical integrity. (See Asada, et al., Biosci.Biotech. Biochem., vol. 61, no. 6, pgs. 1030-1032 (1997).)

Both the M and G sugar residues contain carboxyl groups with pKa'saround 3.5. At physiological pH, the carboxyl groups are ionized,forming long chains of repeating negatively-charge carboxyl groups thatcan hydrogen bond to water to form a thick tissue-adherent gel, e.g.bioadhesive. Alginates also adhere to mucosal surfaces using hydrogenbonding. It is believed that alginates can act as an “artificial mucosa”that protects against both chemical and physical trauma. Due to theinnate mucoadhesive and bioadhesive properties of alginic acid, itshould remain within the vagina for about 12 to 24 hours (or evenlonger) if not removed by the woman.

Alginates have anti-inflammatory capabilities. Specifically, studieshave shown that alginic acid inhibits NFkB activation. NFkB is a proteincomplex that controls the transcription of DNA. (See Jeong, et al.,Clinical and Experimental Allergy, vol. 36, pgs. 785-794 (2006).) NFkBis involved in cellular responses to stimuli such as stress, cytokines,free radicals, ultraviolet irradiation, oxidized LDL, and bacterial orviral antigens. NFkB plays a key role in regulating the immune responseto infection. It is now known that many viruses, including HIV,hepatitis, and herpes, activate NFkB to facilitate host cell infection.By preventing the activation of NFkB and, by extension, preventing theactivation of multiple inflammatory pathways, alginic acid reduces therisk of viral STD infection.

Alginates also inhibit immunoglobulin E-mediated mast celldegranulation. (See Asada, et al., Biosci. Biotech. Biochem., vol. 61,no. 6, pgs. 1030-1032 (1997).) Mast cells play an important role indefense against pathogens. Pathogens can activate mast cells throughstimulation of the immunoglobulin E (IgE) receptor or through patternrecognition receptor, which include toll-like receptors (TLRs), Nod-likereceptors, C-type lectins, and the glycosylphosphatidylinositol-anchoredprotein CD48. Upon activation, mast cells release various mediators thataffect vascular permeability and trigger additional immune responses.The mediators include granule-associated mediators, including histamine,serotonin, heparin, tryptase, chymase, and tumour necrosis factor-alpha,cytokines, and chemokines. In mucosal tissue, which can be the primarysite for HIV infection, activation of mast cells, which leads toincreased vascular permeability and mast cell interaction with T-cellsmay contribute to HIV infectivity. Furthermore, in HIV infection, mastcells may serve as a viral reservoir during latent infection and can bereactivated through TLR-mediated signals. (See Urb M and Sheppard, D C(2012) “The Role of Mast Cells in the Defence against Pathogens.” PLoSPathog 8(4): e1002619. doi:10.1371/journal.ppat. 1002619.)

It has been unexpectedly discovered that not all molecular weights ofalginic acid are affective at blocking inflammation. Specificallyalginates with molecular weights between 20,000 to 400,000 g/mol displaygreater capabilities in inhibiting mast cell degranulation. In oneembodiment, the molecular weight of the alginate may be between 100,000to 375,000 g/mol. In another embodiment, the molecular weight of thealginate may be between 200,000 and 350,000 g/mol. In yet anotherembodiment, the molecular weight of the alginate may be between 290,000and 340,000 g/mol. (See Asada, et al., Biosci. Biotech. Biochem., vol.61, no. 6, pgs. 1030-1032 (1997).)

It has also been unexpectedly discovered that the M:G ratio also affectsthe anti-inflammatory properties of alginic acid. In one embodiment, theM:G ratio may be between 0.25 and 2.0. In another embodiment, the M:Gratio may be between 0.3 and 1.5. In another embodiment, the M:G rationis between 0.5 and 1.2. In yet another embodiment, the M:G ratio is 1.(See Asada, et al., Biosci. Biotech. Biochem., vol. 61, no. 6, pgs.1030-1032 (1997), incorporated by reference herein. Asada describes the0.6 type sodium alginate as having an M:G ratio of 3:7, see secondparagraph of article, however, an M:G ration of 3:7 mathematically is0.43)

As noted above, alginates cross-link in the presence of monovalent anddivalent cations such as sodium, potassium, and calcium. Vaginal fluidsgenerally contain very little amounts of these cations. Semen, on theother hand, is abundant in these cations. Thus, in the presence ofsemen, alginic acid will cross-link to form a matrix that can trap spermand microbes. Trapping sperm and other microbes present in semen willreduce the risk of transmission of STDs. Additionally, alginic acid hasa pH of about 1.5 to 3.5 in an aqueous solution. Though not wishing tobe bound by any particular theory of operation, the naturally low pH ofalginic acid may help in maintaining a healthy vaginal pH (i.e. between3.5 to 5.0). As noted above, a low pH inactivates many STD-causingmicrobes. Furthermore, alginic acid has bioadhesive and mucoadhesiveproperties which will keep the alginic acid-based compositions in placeduring sexual activity. Lastly, alginic acid is generally not absorbedby the body due to its high molecular weight.

Tenofovir

Tenofovir, which includes derivatives, analogues, prodrugs and saltsthereof, belongs to a class of antiretroviral drugs known as nucleotideanalogue reverse transcriptase inhibitors (NtRTIs), which block reversetranscriptase. It has the chemical name1-(6-aminopurin-9-yl)propan-2-yloxymethylphosphonic acid [CAS Registrynumber: 147127-20-6]. Tenofovir is commercially available, such as fromGilead Sciences, Inc., (Foster City, Calif.). The structure of tenofoviris shown below:

Tenofovir is a competitive inhibitor of other naturally occurringnucleotides, and its ultimate biological activity is viral DNA chaintermination. Tenofovir is a novel nucleotide analog with antiviralactivity against both HIV and Hepatitis B. The mechanism of tenofovir issimilar to that of nucleoside analogs, which interfere with reversetranscriptase and prevents translation of viral genetic material intoviral DNA. Unlike the nucleoside analogs, the NtRTIs are chemicallypre-activated with the presence of a phosphate group. Since thephosphorylation step is not necessary, nucleotide analogs canincorporate into viral DNA chain more rapidly than nucleoside analogs.More importantly, this will bypass a viral mechanism of nucleosideresistance.

Acid Buffering Contraceptive

In one embodiment, the specific alginic acid subsets described above canbe part of an acid buffering contraceptive. One non-limiting example ofsuch acid buffering contraceptive is Acidform (also known as Amphora®gel (U.S. Pat. No. 6,706,276, WO 01/66084)), which is a gel that, whenplaced in a body orifice (e.g., the vagina), forms a matrix upon contactwith ejaculate and thus entraps and inactivates spermatozoa and/or STDand STI-causing microbes. In one general embodiment, the acid bufferingcontraceptive contains (1) a matrix-forming compound, (2) a bioadhesivecompound, and (3) lactic acid. Some compounds, such as alginic acid,carrageenan, or chitosan, can act as both the matrix-forming compoundand the bioadhesive compound.

In exemplary embodiments, the Acidform used generally contains (1) about1-10% of one or more matrix-forming compounds, (2) about 1-10% of one ormore bioadhesive compounds, and (3) about 1-10% of lactic acid. In otherembodiments of, the Acidform composition contains (1) about 3-5% of oneor more matrix-forming compounds, (2) about 2.5-6% of one or morebioadhesive compounds, and (3) about 1-7% of lactic acid. In otherembodiments, the Acidform composition contains (1) about 3.5-4.5% of oneor more matrix-forming compounds, (2) about 2.5-3.5% of one or morebioadhesive compounds, and (3) about 1-4% of lactic acid.

In other exemplary embodiment, the Acidform used generally contains (1)about 1-10% of one or more matrix-forming compounds, (2) about 1-10% ofone or more bioadhesive compounds, and (3) about 1-10% of L-lactic acid.In other embodiments, the Acidform composition contains (1) about 3-5%of one or more matrix-forming compounds, (2) about 2.5-6% of one or morebioadhesive compounds, and (3) about 1-7% of L-lactic acid. In otherembodiments, the Acidform composition contains (1) about 3.5-4.5% of oneor more matrix-forming compounds, (2) about 2.5-3.5% of one or morebioadhesive compounds, and (3) about 1-4% of L-lactic acid.

Matrix-forming compounds suitable for use in the present disclosureshould be stable over a wide pH range, especially over the normal acidicpH values found in the vagina. Suitable matrix-forming compoundsinclude, for example, alginic acid, chitosan, gellan gum, poloxamer,carrageenan, iota carrageenan, and the like. The matrix-forming compoundpreferably stays in a non-matrix state until it comes in contact withejaculate. Upon contact with ejaculate, the matrix-forming compoundforms a semisolid matrix that traps sperm and STD-causing microbes sothey cannot migrate through the lower female genital tract. Some matrixforming compounds, such as alginic acid, (which has a pH of about1.5-3.5 in an aqueous solution), may contribute to the acid bufferingcapabilities of the acid buffering contraceptive. Furthermore, somematrix-forming compounds may also contribute to the bioadhesive natureof the acid buffering contraceptive. In some embodiments, thematrix-forming compound and bioadhesive compound are the same.

Bioadhesive compounds suitable for use in the present disclosureinclude, for example, xanthan gum, carrageenan, iota carrageenan,alginic acid, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,sodium carboxymethyl cellulose, chitosan, polycarbophil, carbopol, andthe like. In at least one embodiment, the bioadhesive compound isxanthan gum, a high molecular weight polysaccharide gum containingD-glucosyl, D-mannosyl, and D-glucosyluronic acid residues and varyingproportions of 0-acetyl and pyruvic acid acetal. The primary structureis a cellulose backbone with trisaccharide side chains; the repeatingunit is a pentasaccharide. Generally, the molecular weight is greaterthan about 106 g/mole.

The acid buffering contraceptive further comprises lactic acid or otherbuffering agents that act to maintain the pH of the vagina within itsnormal acidic range (i.e., a pH of less than about 5 and more preferablyin the range of about 3.5 to about 4.5) even in the presence of normalamounts of ejaculate. Besides lactic acid, suitable buffering agentsinclude, but are not limited to, for example, citric acid, potassiumacid tartrate, benzoic acid, alginic acid, sorbic acid, fumaric acid,ascorbic acid, stearic acid, oleic acid, tartaric acid, potassiumbitartrate, benzoic acid, edetic acid ethylenediaminetetracetic acid,acetic acid, malic acid, and the like. The acids may be added as freeacids, hydrates, or pharmaceutically acceptable salts. Of course, thefree acids can be converted to the corresponding salts in situ (i.e.,within the vagina). In various exemplary embodiments, several bufferingagents are included in the Acidform composition to provide increasedbuffering capacity. Alginic acid, of course, can function as both amatrix-forming agent and a buffering agent. Since alginic acid will notbe absorbed by the body, its acid buffering effect will be longerlasting as compared to the other buffering agents which may be absorbedby the body.

Accordingly, as discussed above, lactic acid or other suitable bufferingagents may be used to maintain the pH of the vagina within its normalacidic range after application (i.e., a pH of less than about 5 and morepreferably in the range of about 3.5 to about 4.5). In particular, ithas been discovered that lactic acid significantly increases themicrobicidal potency in relation to other natural vaginal defensemechanisms, such as hydrogen peroxide. This feature was previouslyunknown to those of skill in the art, and the inventors of the presentdisclosure have surprisingly found that the acid bufferingcontraceptive, when formulated using lactic acid as a buffering agent,possesses significantly greater microbicidal activity than formulationsthat do not use lactic acid as a buffering agent.

Specifically, the presence of lactic acid results in greaterinactivation of microbes, including viruses, in comparison to compoundssuch as hydrogen peroxide or acetic acid at equivalent pH. The mechanismof action by which lactic acid increases microbicidal potency isbelieved to be the disruption of the cell membranes of gram-negativebacteria, and also acts to inactivate HIV and HSV-2.

More specifically, lactic acid has two isomers, one is known asL-(+)-lactic acid or (S)-lactic acid and the other is D-(−)-lactic acidor (R)-lactic acid. Recent discovery has shown that the L form of lacticacid is more potent in inactivating HIV than D or racemic lactic acid.While the precise mechanism of how L-lactic acid inactivates HIV isunknown, the stereochemical dependent activity suggests that it acts onproteins. (Purcell et al., AIDS Res Hum Retroviruses. 2012 November;28(11):1389-96.)

Lactic acid is produced by lactic acid bacteria such as Lactobacillusspecies. However, lactic acid bacteria generally produce both D and Llactic acid. Furthermore, lactic acid bacteria can be difficult to grow.Recombinant methods can be used to specifically manufacture L-lacticacid using hosts that easier to grow such as yeast or Escherichia coli.(Ishida et al., Appl Environ Microbiol. 2005 April; 71(4): 1964-1970 andDien et al., J Ind Microbiol Biotechnol. 2001 October; 27(4):259-64.)Alternatively, purified L-lactic acid can be purchased from establishedchemical suppliers such as Sigma-Aldrich® (St. Louis, Mo.).

In one embodiment the acid buffering contraceptive is further describedas follows: the matrix-forming compound is alginic acid; the bioadhesivecompound is xanthan gum, and/or hydroxycellulose, and/or carrageenan;lactic acid is used or is substituted by citric acid, benzoic acid orpotassium acid tartrate; glycerol is included as a humectant; benzoicacid is used as a preservative; and water is the pharmaceuticallyacceptable carrier. In another embodiment, the composition containsxanthan gum, alginic acid, lactic acid, citric acid, benzoic acid,potassium bitartrate, glycerol, and water. In another embodiment, thelactic acid is L-lactic acid.

In another embodiment, the acid buffering contraceptive includes alginicacid, carrageenan, and lactic acid. In another embodiment, the acidbuffering contraceptive includes alginic acid, carrageenan, lactic acid,citric acid, benzoid acid, potassium bitartrate, glycerol, and water. Inanother embodiment, the carrageenan is iota carrageenan. In anotherembodiment, the lactic acid is L-lactic acid.

Pharmaceutically Acceptable Carrier

In one embodiment, the pharmaceutical carrier is water. Otherpharmaceutically acceptable carriers that are suitable for vaginaldelivery are well known and can be used in place of water. One exampleof a suitable pharmaceutically acceptable carrier is petrolatum, such aswhite petrolatum.

Optional Ingredients

Additional optional excipients may be used in the compositions of thepresent disclosure such as buffering agents, thickeners, humectants, andpreservatives. Suitable buffering agents include, but are not limitedto, for example, lactic acid, citric acid, potassium acid tartrate,potassium bitartrate, benzoic acid, sorbic acid, fumaric acid, ascorbicacid, stearic acid, oleic acid, tartaric acid, edetic acidethylenediaminetetracetic acid, acetic acid, malic acid, and the like.Suitable thickeners include, but are not limited to, for example,xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,sodium carboxymethyl cellulose, chitosan, polycarbophil, carbopol,gellan gum, poloxamer, carrageenan, iota carrageenan, and the like.Suitable humectants include, but are not limited to, for example,glycerol, polyethylene glycols, propylene glycols, sorbitol, triacetin,and the like. In one exemplary embodiment, glycerol is used to preventthe formation of a dry film on the gel when placed within the vagina.Glycerol may also act as a lubricant. Additionally, the compositions mayalso include a preservative. Suitable preservatives include, but are notlimited to, for example, benzoic acid, sodium benzoate, methylparaben,ethylparaben, butylparaben, propylparaben, benzylalkonium chloride,phenylmercuric nitrate, chlorhexidine, and the like. In one exemplaryembodiment, benzoic acid is used and may also contribute to thebuffering capacity of the Acidform gel.

Optional ingredients also include agents that enhance drug solubility,permeability and absorption. Non-limiting examples include pamoic acid(also called “embonic acid”) and salts and esters thereof.

Formulation

The pharmaceutical composition may be in the form of a gel, asemi-solid, a cream, and/or a lotion. Generally, the alginic acid-basedmicrobicide may be administered as a topical ointment applied to thelining of the vagina and/or cervix and/or rectum, which can beaccomplished as a gel, cream, lotion, non-aqueous or aqueous solutionused to flush the vaginal or rectal cavity, and/or a vaginal or rectalsuppository. In other embodiments, the alginic acid-based microbicidecomposition may be administered in a spray formulation. In addition, thealginic acid-based microbicide compositions may be delivered usingmicrobicide-impregnated diaphragms and female and male condoms.

Furthermore, in addition to the alginic acid-based microbicidecompositions disclosed herein, the balance of the compositions, i.e.,typically from about 0-10% weight, or from about 0.1-5% weight, or fromabout 0.1-3% weight, may optionally comprise one or more cosmeticingredients. Such cosmetic ingredients are known to those skilled in theart and are often referred to in the art as diluents, solvents, andadjuvants. Typically, cosmetic ingredients include, for example; water,ethyl alcohol, isopropyl alcohol, glycerin, glycerol propylene glycol,sorbitol, and other high molecular weight alcohols. In addition, thecompositions may contain minor amounts of other additives, such as, forexample; stabilizers, surfactants, menthol, eucalyptus oil, otheressential oils, fragrances, and the like. The selection and amounts ofcosmetic ingredients, other additives, and blending procedures can becarried out in accordance with techniques well-known in the art.

Method of Manufacture

Tenofovir is an off-white powder with a molecular weight of 287.2(anhydrous) or 305 (as monohydrate). It is not water soluble at anacidic pH and thus is not easily combinable with alginic acid. Thepresent disclosure provides various manufacturing methods for combiningtenofovir with alginic acid.

The final formulation should be viscous enough so that it remains inplace without the use of physical devices. Compositions that are toothin will leak out and compositions that are too thick will be difficultto use properly, (e.g. smear to cover the vagina and cervix).Additionally, during use, dilution is expected to occur due to, forexample, the presence of vaginal fluid and exposure to semen. Theviscosity of the formulations described herein may be between20,000-200,000 centipoise (cP) when the torque percentage is about 20%.Alternatively, the viscosity maybe between 30,000-150,000 cP.

The methods disclosed herein generally involve three steps. In the firststep, tenofovir is dissolved in a basic solution. In the second step,the pH is brought to below neutral. In the third step, alginic acid isadded. Optional ingredients, such as thickeners, humectants, orpreservatives may be added during any of the steps so long as theoptional ingredients do not affect the overall pH of the solutions inthe various steps.

In one embodiment, the first step combines water, tenofovir, and sodiumhydroxide. The second step adds lactic acid. The third step adds alginicacid. In another embodiment, the first step combines water, tenofovir,and sodium hydroxide. The second step adds lactic acid. The third stepadds alginic acid and iota carrageenan. In yet another embodiment, thefirst step combines water, tenofovir, and sodium hydroxide. The secondstep adds L-lactic acid. The third step adds alginic acid and iotacarrageenan.

The above-described method can also be used for incorporating thealginic acid-based microbicide into an acid buffering contraceptive. Inone embodiment, the first step combines water, tenofovir, and sodiumhydroxide. The second step adds lactic acid. The third step adds alginicacid and xanthan gum. Alternatively, the first step combines water,tenofovir, and sodium hydroxide. The second step adds citric acid andlactic acid. The third step adds alginic acid and xanthan gum. Inanother alternate method, the first step combines water, tenofovir, andsodium hydroxide. The second step adds benzoic acid, citric acid, andlactic acid. The third step adds alginic acid and xanthan gum. In yetanother alternate method, the first step combines water, tenofovir, andsodium hydroxide. The second step adds benzoic acid, citric acid,potassium bitartrate, and lactic acid. The third step adds alginic acidand xanthan gum. In yet another alternate method, the first stepcombines water, tenofovir, and sodium hydroxide. The second step addsbenzoic acid, citric acid, potassium bitartrate, and lactic acid. Thethird step adds alginic acid, glycerin, and xanthan gum.

In another embodiment, the first step combines water, tenofovir sodiumhydroxide, and benzoic acid. The second step adds lactic acid. The thirdstep adds alginic acid and xanthan gum. Alternatively, the first stepcombines water, tenofovir sodium hydroxide, and benzoic acid. The secondstep adds citric acid and lactic acid. The third step adds alginic acidand xanthan gum. In another alternate method, the first step combineswater, tenofovir sodium hydroxide, and benzoic acid. The second stepadds potassium bitartrate, citric acid, and lactic acid. The third stepadds alginic acid and xanthan gum. In yet another alternate method, thefirst step combines water, tenofovir sodium hydroxide, and benzoic acid.The second step adds potassium bitartrate, citric acid, and lactic acid.The third step adds alginic acid, glycerin, and xanthan gum.

In yet another embodiment, the first step combines water, tenofovir,sodium hydroxide, benzoic acid, and potassium bitartrate. The secondstep adds lactic acid. The third step adds alginic acid and xanthan gum.Alternatively, the first step combines water, tenofovir, sodiumhydroxide, benzoic acid, and potassium bitartrate. The second step addscitric acid and lactic acid. The third step adds alginic acid andxanthan gum. In another alternate method, the first step combines water,tenofovir, sodium hydroxide, benzoic acid, and potassium bitartrate. Thesecond step adds citric acid and lactic acid. The third step addsalginic acid, glycerin, and xanthan gum.

In yet another embodiment, the first step combines water, tenofovir, andsodium hydroxide. The second step adds lactic acid. The third step addsalginic acid and iota carrageenan. Alternatively, the first stepcombines water, tenofovir, and sodium hydroxide. The second step addscitric acid and lactic acid. The third step adds alginic acid and iotacarrageenan. In another alternate method, the first step combines water,tenofovir, and sodium hydroxide. The second step adds benzoic acid,citric acid, and lactic acid. The third step adds alginic acid and iotacarrageenan. In yet another alternate method, the first step combineswater, tenofovir, and sodium hydroxide. The second step adds benzoicacid, citric acid, potassium bitartrate, and lactic acid. The third stepadds alginic acid and iota carrageenan. In yet another alternate method,the first step combines water, tenofovir, and sodium hydroxide. Thesecond step adds benzoic acid, citric acid, potassium bitartrate, andlactic acid. The third step adds alginic acid, glycerin, and iotacarrageenan.

In yet another embodiment, the first step combines water, tenofovirsodium hydroxide, and benzoic acid. The second step adds lactic acid.The third step adds alginic acid and iota carrageenan. Alternatively,the first step combines water, tenofovir sodium hydroxide, and benzoicacid. The second step adds citric acid and lactic acid. The third stepadds alginic acid and iota carrageenan. In another alternate method, thefirst step combines water, tenofovir sodium hydroxide, and benzoic acid.The second step adds potassium bitartrate, citric acid, and lactic acid.The third step adds alginic acid and iota carrageenan. In yet anotheralternate method, the first step combines water, tenofovir sodiumhydroxide, and benzoic acid. The second step adds potassium bitartrate,citric acid, and lactic acid. The third step adds alginic acid,glycerin, and iota carrageenan.

In yet another embodiment, the first step combines water, tenofovir,sodium hydroxide, benzoic acid, and potassium bitartrate. The secondstep adds lactic acid. The third step adds alginic acid and iotacarrageenan. Alternatively, the first step combines water, tenofovir,sodium hydroxide, benzoic acid, and potassium bitartrate. The secondstep adds citric acid and lactic acid. The third step adds alginic acidand iota carrageenan. In another alternate method, the first stepcombines water, tenofovir, sodium hydroxide, benzoic acid, and potassiumbitartrate. The second step adds citric acid and lactic acid. The thirdstep adds alginic acid, glycerin, and iota carrageenan.

Methods of Use

In exemplary embodiments, the present disclosure involves the topicalapplication of alginic acid-based antimicrobial compositions asdescribed herein. In the context of the present disclosure, it is to beunderstood that the term “topical application” includes application tothe body cavities as well as to the skin. Thus, for example, theaforementioned compositions are applied to a body cavity such as thevagina, anus, rectum or mouth. Furthermore, the topical application maybe carried out before, during or after intercourse, or alternatively,carried out independent from intercourse.

It is to be understood that the alginic acid-based antimicrobialcompositions of the present disclosure may be delivered to the vagina ofa mammal by any means known to those skilled in the art. Typical formsfor delivery of the compositions include, for example; creams, lotions,gels, foams, intervaginal devices such as sponges and suppositories, andfilms. In addition, the alginic acid-based antimicrobial compositionsmay be used as personal care products, such as, for example, condomlubricants, and the like. Such lubricants may comprise commonly knowningredients such as, for example: humectants, e.g., glycerin, sorbitol,mannitol, glycols and glycol ethers; buffers, e.g., glucono-d-lactone;germicides or bactericides, e.g., chlorhexidine gluconate;preservatives, e.g., methylparaben; viscosifiers, e.g., hydroxyethylcellulose, etc.; other adjuvants, e.g., colors and fragrances; inaddition to the compositions of the present disclosure. Those skilled inthe art will recognize that the physical properties, e.g., viscosity, ofsuch delivery forms may vary widely. For example, the viscosity of a gelform of the composition of the present disclosure, e.g. 150,000centipoise, may be substantially higher than the viscosity of lotionform of the composition of the present disclosure, e.g., 100 centipoise.Further details concerning the materials, ingredients, proportions andprocedures of such delivery forms can be selected in accordance withtechniques well-known in the art.

In various embodiments, the alginic acid-based antimicrobialcompositions of the present disclosure are preferably administered tothe vagina of the mammal in a dosage which is effective to reduce therisk of STD transmission. Typical dosages range between about 1-10grams, or between 3-7 grams, or between 4-6 grams of the composition. Invarious embodiments, the alginic acid-based antimicrobial compositionsdisclosed can be applied using a device or applicator such as asuppository, a sponge, a swab, a brush, or a syringe. In otherembodiments, the alginic acid-based antimicrobial compositions disclosedcan be applied to a barrier device such as a sponge, a condom or adiaphragm. In one embodiment, the alginic acid-based antimicrobialcompositions disclosed can be in pre-filled single use devices such as apre-filled squeezable tube or pre-filled syringe.

It will be readily apparent to those skilled in the art that othercompounds functioning as precursors, analogs and derivatives such assalts and esters of the present compounds can be utilized.

The disclosure set forth above is provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use embodiments of the compositions and methods, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes (for carrying outthe disclosure that are obvious to persons of skill in the art) areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference in their entirety asif each such publication, patent or patent application were specificallyand individually indicated to be incorporated herein by reference.

EXAMPLES

The following formulations use a subset of alginic acid that has amolecular weight between 20,000 and 400,000 and an M:G ration between0.25 and 2.0.

Example 1: Formulations for Alginic Acid-Based Microbicide

In its simplest form, the alginic acid-based microbicide containsalginic acid, tenofovir, and water. In another form, alginic acid, iotacarrageenan, tenofovir, and water are combined. For any of theformulations mentioned herein, pamoic acid is optionally added toenhance the solubility, permeability, or absorption of tenofovir.

The formulations are made by dissolving tenofovir in a basic solution ofwater and sodium hydroxide. The pH is brought to below neutral and thealginic acid is added. Iota carrageenan, if being used, is added before,at the same time, or after the alginic acid.

Example 2: Formulations for Alginic Acid-Based Microbicide andContraceptive

In its simplest form, the alginic acid-based microbicide andcontraceptive contains alginic acid, tenofovir, lactic acid and water.In another form, alginic acid, xanthan gum, tenofovir, lactic acid, andwater are combined. In another form, alginic acid, iota carrageenan,tenofovir, lactic acid, and water are combined. In another form, alginicacid, xanthan gum, tenofovir, lactic acid, citric acid, benzoic acid,potassium bitartrate, glycerin, and water are combined. In another form,alginic acid, iota carrageenan, tenofovir, lactic acid, citric acid,benzoic acid, potassium bitartrate, glycerin, and water are combined.For any of the formulations mentioned herein, pamoic acid is optionallyadded to enhance the solubility, permeability, or absorption oftenofovir.

The formulations are made by dissolving tenofovir in a basic solution ofwater and sodium hydroxide. The pH lowering ingredients, such as citricacid, lactic acid, and alginic acid are added next. Thickening agents,such as xanthan gum and iota carrageenan are added last. Otheringredients that do not affect the pH, such as glycerin, benzoic acid,and potassium bitartrate, can be added at any stage. The final pH shouldbe similar to the normal vaginal pH range, such as between about 2 andabout 5 or between about 2 and about 4, or between about 3 and about 4.

Example 3: Clinical Trials for an Acid Buffering Contraceptive, AmphoraGel

In this example, clinical trials were conducted to evaluate thecontraceptive capabilities of an acid buffering contraceptive, Amphoragel. Amphora gel was compared to Conceptrol® vaginal gel (RevivePersonal Products Company, Madison, N.J.) in a non-inferiority study.Conceptrol vaginal gel is a topical gel containing 4% nonoxynol-9 in a2.5 mL volume of gel. Healthy women between the ages of 18-35 wereenlisted in the clinical trial, half were given Amphora gel (5 mL doseper use) and the other half were given Conceptrol gel (2.5 mL dose peruse). Women were instructed to vaginally insert the gel up to 2 hoursbefore having heterosexual intercourse. Both gels were administered overa period of 7 menstrual cycles and a subset of users continued usingAmphora gel for an additional 6 cycles. At the end of the evaluationperiod, a subset of women underwent colposcopy to detect lesions in thevaginal and cervix and testing to measure any changes in the vaginalflora.

The results indicated that Amphora gel was not inferior to Conceptrolvaginal gel for contraceptive purposes using Kaplan-Meier statisticalanalysis. Amphora gel was well tolerated and had no significant adverseevent. The incidence of bacterial vaginosis, urinary tract infection,and yeast infections were similar between the two gels. Further, therewere no reports of leakage of the Amphora gel and the women seemed toprefer Amphora gel.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use embodiments of the compositions, and are not intended to limitthe scope of what the inventors regard as their invention. Modificationsof the above-described modes (for carrying out the invention that areobvious to persons of skill in the art) are intended to be within thescope of the following claims.

All references cited herein are hereby incorporated by reference.

What is claimed is:
 1. A composition for inhibiting inflammation anddisease comprising 1-(6-aminopurin-9-yl)propan-2-yloxymethylphosphonicacid (tenofovir) or a physiologically functional derivative thereof,alginic acid, and an aqueous based carrier; wherein the alginic acid hasan average molecular weight between 20,000 to 400,000; and wherein thealginic acid has a ratio of mannuronate:guluronate residues between 0.25to 2.0.